Figure 4. Hst/Py-sorted HSCs display an absolute but transient S/G₂/M engraftment defect.

(A) Ter119^– 14.5-dpc FL cells in G₁/S/G₂/M were fractionated into their component G₁ and S/G₂/M subsets, leaving a slight separation between them. Aliquots of the sorted subsets were then stained with PI as well as with Hst/Py (data not shown). The sorted cells were cultured for 6 hours and then stained again with PI. We found that during this 6-hour culture period, approximately one-third of the cells originally in G₁ had progressed into S/G₂/M, and a similar proportion of the cells originally in S/G₂/M had progressed into G₁. (B) CRUs per 10⁵ initial Ter119^– FL cells for G₁ and S/G₂/M fractions before and after 6 hours in culture. There was a 3.5-fold loss of CRUs when G₁ cells were cultured for 6 hours, but no loss when the cultured cells were re-sorted for G₁ cells (P = 0.36). Conversely, we detected a greater than 65-fold increase in the number of CRUs detected when CRUs in S/G₂/M were cultured and a greater than 128-fold increase when the cultured cells were sorted for G₁ cells. IF, intrafemoral. Values are mean ± SEM of results from at least 3 experiments. *P < 0.01, **P < 0.001 versus respective cell types before culture.